Chapter 6 (Cell Kinetics and Fermenter Design-127-175) PDF

6
Cell Kinetics and Fermenter Design
6.1 INTRODUCTION
Understanding the growth kinetics of microbial, animal, or plant
cells is important for the design and operation of fermentation
systems employing them. Cell kinetics deals with the rate of cell
growth and how it is affected by various chemical and physical
conditions.
Unlike enzyme kinetics discussed in Chapter 2, cell kinetics is the
result of numerous complicated networks of biochemical and
chemical reactions and transport phenomena, which involves
multiple phases and multicomponent systems. During the course of
growth, the heterogeneous mixture of young and old cells is
continuously changing and adapting itself in the media environment
which is also continuously changing in physical and chemical
conditions. As a result, accurate mathematical modeling of growth
kinetics is impossible to achieve. Even with such a realistic model,
this approach is usually useless because the model may contain many
parameters which are impossible to determine.
Therefore, we must make assumptions to be able to arrive at
simple models which are useful for fermenter design and
performance predictions. Various models can be developed based on
the assumptions concerning cell 'components and population as
shown in Table 6.1 (Tsuchiya et al., 1966). The simplest model is the
unstructured, distributed rnodel which is based on the following two
assumptions:
1. Cells can be represented by a single component, such as cell
mass, cell number, or the concentration of protein, DNA, or
RNA. This is true for balanced growth, since a doubling of cell
mass for balanced growth is accompanied by a doubling of all
other measurable properties of the cell population.
2. The population of cellular mass is distributed uniformly
throughout the culture. The cell suspension can be regarded as
a homogeneous solution. The heterogeneous nature of cells can
be ignored. The cell concentration can be expressed as dry
weight per unit volume.
128 Fundamentals of Biochemical Engineering
Table 6.1 Various Models for Cell Kinetics
Cell Components
Population Unstructured Structured
Distributed Cells are represented by a Multiple cell components,
single component,which is uniformly distributed
uniformly distributed throughout the culture
throughout the culture interact with each others
Segregated Cells are represented by a Cells are composed of
single component, but they multiple components and
form a heterogeneous mixture form a heterogeneous
mixture
Besides the assumptions for the cells, the medium is formulated so

that only one component may be limiting the reaction rate. All other
components are present at sufficiently high concentrations, so that
minor changes do not significantly affect the reaction rate.
Fermenters are also controlled so that environmental parameters
such as pH, temperature, and dissolved oxygen concentration are
maintained at a constant level.
In this chapter, cell kinetic equations are derived from the
unstructured, distributed model, and those equations are applied for
the analysis and design of ideal fermenters. More realistic models
which consider the multiplicity of cell components, structured
model, are introduced at the end of the chapter.
6.2 DEFINITIONS
First, let us define the terminologies we use for microbial growth. If
we mention the cell concentration without any specification, it can
have many different meanings. It can be the number of cells, the wet
cell weight, or the dry cell weight per unit volume. In this text, the
following nomenclature is adopted:
Cx Cell concentration, dry cell weight per unit volume
CN Cell number density, number of cells per unit volume
p Cell density, wet cell weight per unit volume of cell mass
Accordingly, the growth rate can be defined in several different
ways, such as:
dC x I dt Change of dry cell concentration with time
rx Growth rate of cells on a dry weight basis
dCNldt Change of cell number density with timernGrowth rate of
cells on a number basis
8 Division rate of cells on a number basis, d logz CNI dt
eel/Kinetics and Fl'rJlll'nter Design 129
It appears that dCx/dt and rx are always the same, but this is not
true. The former is the change of the cell concentration in a fermenter,
which may include the effect of the input and_ output flow rates, cell
recycling, and other operating conditions of a fermenter. The latter is
the actual growth rate of the cells. The two quantities are the same
only for batch operation.
The growth rate based on the number of cells and that based on cell
weight are not necessarily the same because the average size of the
cells may vary considerably from one phase to another. When the
mass of an individual cell increases without division, the' growth rate
based on cell weight increases, while that based on the number of
cells stays the same. However, during the exponential growth period,
which is the phase that we are most interested in from an engineer's
point of view, the growth rate based on the cell number and that
based on cell weight can be assumed to be proportional to each other.
Sometimes, the growth rate can be confused with the division rate,
which is defined as the rate of cell division per unit time. If all of the
cells in a vessel at time t = 0 (C N = CN ) have divided once after a
certain period of time, the cell populcftion will have increased to
CN x 2. If cells are divided n times after the time t, the total number of
cel~s will be
C N =C Np X2 N (6.1)
and the average division rate IS
8=~ (6.2)
Since N = log2CN - log2CN according to Eq. (6.1), the average
division rate is 0
- 1
8 =-- (log2 eN log2 eN ) (6.3)
t 0
and the division rate at time t is
8 = dlog z CN (6.4)
dt
Therefore, the growth rate defined as the change of cell number
with time is the slope of the CN versus t curve, while the division rate
is the slope of the log2CN versus t curve. As explained later, the
division rate is constant during the exponential growth period, while
the growth rate is not. Therefore, these two terms should not be
confused with each other.
6.3 GROWTH CYCLE FOR BATCH CULTIVATION

If you inoculate unicellular microorganisms into a fresh sterilized
medium and measure the cell number density with respect to time
and plot it, you may find that there are six phases of growth and
death, as shown in Figure 6.1. They are:
1. Lag phase: A period of time when the change of cell number is
zero.
2. Accelerated growth phase: The cell number starts to increase
and the division rate increases to reach a maximum.
3. Exponential growth phase: The cell number increases
exponentially as the cells start to divide. The growth rate is
increasing during this phase, but the division rate which is
proportional to dlnCN1 / dt, is constant at its maximum value, as
illustrated in Figure 6.1.
4. Decelerated growth phase: After the growth rate reaches a
maximum, it is followed by the deceleration of both growth rate
and the division rate.
5. Stationary phase: The cell population will reach a maximum
value and will not increase any further.
6. Death phase: After nutrients available for the cells are
depleted, cells will start to die and the number of viable cells
will decrease.
6.3.1 Lag Phase

The lag phase (or initial stationary, or latent) is an initial period of
cultivation during which the change of cell nU:f!lber is zero or
negligible. Even though the cell number does not increase, the cells
may grow in size during this period.
The length of this lag period depends on many factors such as the
type and age of the microorganisms, the size of the inoculum, and
culture conditions.
The lag usually occurs because the cells must adjust to the new
medium before growth can begin. If microorganisms are inoculated
from a medium with a low nutrient concentration to a medium with a
high concentration, the length of the lag period is usually long. This
is because the cells must produce the enzymes necessary for the
metabolization of the available nutrients. If they are moved from a
high to a low nutrient concentration, there is usually no lag phase.
Another important factor affecting the length of the lag phase is
the size of the inoculum. If a small amount of cells are inoculated into
a large volume, they will have a long lag phase. For large-scale
operation of the cell culture, it is our objective to make this lag phase
as short as possible. Therefore, to inoculate a large fermenter, we
need to have a series of progressively larger seed tanks to minimize
the effect of the lag phase.
Cell Kinetics and Fermenter Design 131
12
A I B : C · 0:
~---
E F
1.5
1.0
CN X 10-5
0.5
0
0.3
din CN 0.2
(ft 0.1
0
-0.1
-0.2
Fig. 6.1 Typical growth curve of unicellular organisms: (A) lag
phase; (B) accelerated growth phase; (C) exponential
growth phase; (0) decelerated growth phase;
(E) stationary phase; (F) death phase.
At the end of the lag phase, when growth begins, the division rate
increases gradually and reaches a maximum value in the exponential
growth period, as shown by the rising inflection at B in Figure 6.1.
This transitional period is commonly called the accelerated growth
phase and is often included as a part of the lag phase.
6.3.2 Exponential Growth Phase

In unicellular organisms, the progressive doubling of cell number
results in a continually increasing rate of growth in the population. A
bacterial culture undergoing balanced growth mimics a first-order
autocatalytic chemical reaction (Carberry, 1976; Levenspiel, 1972).
Therefore, the rate of the cell population increase at any particular
time is proportional to the number density (eN) of bacteria present at
that time:
(6.5)
where the constant J.1 is known as the specific growth rate [hr-1j. The
specific growth rate should not be confused with the growth rate,
which has different units and meaning. The growth rate is the change
of the cell number density with time, while the specific growth rate is
1 dCN ' dlnC N
J1 = C at = dt (6.6)
N
which is the change of the natural log of the cell number density
with time. Comparing Eq. (6.4) and Eq. (6.6) shows that
J1 = dlnC N = In 2 (dlogC N )
= 8ln 2 (6.7)
dt dt
Therefore, the specific growth rate J1 is equal to In2 times of the
division rate, 8.
If J1 is constant with time during the exponential growth period,
Eq. (6.5) can be integrated from time t 1 to t as
J CN
C
dCN =
CN
Jtt J1 dt
o
(6.8)
NO
to give
CN = CNo exp [J1 (t - to)] (6.9)
where CN is the cell number concentration at t 1 when the
exponential growth starts. Eq. (6.9) shows the increase of the number
of cells exponentially with respect to time.
The time required to double the population, called the doubling
time (t d ), can be estimated from Eq. (6.9) by setting CN = 2C NO and
t 1 = 0 and solving for t:
td = In2 = 1 (6.10)
J.1 8
The doubling time is inversely proportional to the specific growth
rate and is equal to the reciprocal of the division rate.
1 This equation has the same form as the rate equation for an enzyme catalyzed reaction,
the Michaelis-Menten equation:
r Cs
rp =max
---
K M +C s
and also as the Langmuir adsorption isotherm:
(J=~
K+C A
where (J is the fraction of the solid surface covered by gas molecules and K is the
reciprocal of the adsorption equilibrium constant.
6.3.3 Factors Affecting the Specific Growth Rate

Substrate Concentration: One of the most widely employed
expressions for the effect of substrate concentration on J.1 is the Monod
equation, which is an empirical expression based on the form of
equation normally associated with enzyme kinetics or gas
adsorption: 1
Jl = JlmaxCs (6.11)
Ks +Cs
where Cs is the concentration of the limiting substrate in the
medium and Ks is a system coefficient. This relationship is shown
graphically in Figure 6.2. The value of Ks is equal to the concentration
of nutrient when the specific growth rate is half of its maximum value
(J.1max)
While the Monod equation is an oversimplification of the
complicated mechanism of cell growth, it often adequately describes
fermentation kinetics when the concentrations of those components
which inhibit the cell growth are low.
According to the Monod equation, further increase in the nutrient
concentration after J.1 reaches J.1max does not affect the specific growth
rate, as shown in Figure 6.2. However, it has been observed that the
specific growth rate decreases as the substrate concentration is
increased beyond a certain level.
1.2 ~-----------.
It' Jlmax
0.8 t - = - - - - - - - - - - - - - i
Ks
o ¥
2 4 6
Csx 104 , M
Fig. 6.2 Dependence of the specific growth rate on the

concentration of the growth limiting nutrient: ,
1
Ks = 0.22 x 1(14 molll.
J.1max = 0.935 hr- ,
Several other models have been proposed to improve the Monod

equation. They are:
(6.12)
Ji = Jimax[l- exp(-Cs/Ksl (6.13)
(6.14)
(6.15)
If several substances can limit the growth of a microorganism, the

following model can be employed
C C
Ji = J.lm 1 2 (6.16)
ax K1 + C1 K 2 + C2
If the limiting nutrient is the energy source for the culture, a certain
amount of substrate can be used for purposes other than growth.
Some models include a term, ke, which accounts for the maintenance
of cells as
- JimaxCs - k (6.17)
Ji - e
Ks +Cs
When Cs is so low that the first term of the right-hand side of
Eq. (6.17) is less than or equal to ke, the specific growth rate is equal to
zero. These alternative models give a better fit for the growth of
certain microorganisms, but their algebraic solutions are more
difficult than for the Monod equation.
Product Concentration: As cells grow they produce metabolic by-
products which can accumulate in the medium. The growth of
microorganisms is usually inhibited by these products, whose effect
can be added to the Monod equation as follows:
J.1 -- Ks + C J( K K
(C J
s p
(6.18)
Jimax
s
+c
p p
or
"
/1-
-
Jimax (K C+C J( 1 -C
S
s
5
C
) Pm
p
(6.19)
The preceding equations both describe the product inhibition

fairly well. The term C Pm is the maximum product concentration
above which cells cannot grow due to product inhibition.
Other Conditions: The specific growth rate of microorganisms is
also affected by medium pH, temperature, and oxygen supply. The
optimum pH and temperature differ from one microorganism to
another.
6.3.4 Stationary and Death Phase

The growth of microbial populations is normally limited either by the
exhaustion of available nutrients or by the accumulation of toxic
products of metabolism. As a consequence, the rate of growth
declines and growth eventually stops. At this point a culture is said to
be in the stationary phase. The transition between the exponential
phase and the stationary phase involves a period of unbalanced
growth during which the various cellular components are
synthesized at unequal rates. Consequently, cells in the stationary
phase have a chemical composition different from that of cells in the
exponential phase.
The stationary phase is usually followed by a death phase in which
the organisms in the population die. Death occurs either because of
the depletion of the cellular reserves of energy, or the accumulation of
toxic products. Like growth, death is an exponential function. In
some cases, the organisms not only die but also disintegrate, a
process called lysis.
6.4 STIRRED-TANK FERMENTER
As defined in Chapter 2, a bioreactor is a device within which
biochemical transformations are caused by the action of enzymes or
living cells. The bioreactor is frequently called a fermenter whether
the transformation is carried out by living cells or in vivo cellular
components (enzymes). However, in this text, we call the bioreactor
employing living cells a fermenter to distinguish it from the bioreactor
which employs enzymes, the enzyme reactor. In laboratories, cells are
usually cultivated in Erlenmeyer flasks on a shaker. The gentle
shaking in a shake flask is very effective to suspend the cells, to
enhance the oxygenation through the liquid surface, and also to aid
the mass transfer of nutrients without damaging the structure of cells.
For a large-scale operatiorL, the stirred-tank fermenter (STF) is the
most widely used design in industrial fermentation. It can be
employed for both aerobic or anaerobic fermentation of a wide range
of cells including microbial, animal, and plant cells. Figure 6.3 shows
a diagram of a fermenter used for penicillin production (Aiba et aI.,
1973). The mixing intensity can be varied widely by choosing a
suitable impeller type and by varying agitating speeds. The
mechanical agitation and aeration are effective for the suspension of
cells, oxygenation, mixing of the medium, and heat transfer. The STF
can be also used for high viscosity media. It was one of the first large-
scale fermenters developed in the pharmaceutical industries. Its
performance and characteristics are extensively studied. Since a
stirred-tank fermenter is usually built with stainless steel and
operated in mild operating conditions, the life expectancy of the
fermenter is also long.
The disadvantage of the STF stems from its advantages. The

agitator is effective in mixing the fermenter content, but it consumes
a large amount of power and can damage a shear-sensitive cell
system such as mammalian or plant cell culture. Fluid shear in
mixing is produced by velocity gradients from tangential and radial
velocity components of fluids leaving the impeller region. The
velocity profile of a regular flat-bladed, disk turbine is parabolic in
shape, with the highest observed velocity occurring at the centerline
of each blade. Moving away from the centerline, the resultant
velocity decreases by 85 percent at one blade-width distance above or
below, creating a high shear region (Bowen, 1986). As the blade
width-to-diameter ratio increases, the velocity profile becomes a
more blunt, less parabolic shape, which yields a lower amount of
shear due to a more gradual velocity gradient. Therefore, by
increasing the blade width, a STF can be employed successfully in
cultivating animal cells (Feder and Tolbert, 1983) or plant cells
(Hooker et al., 1990).
Foam
Bam
Heating cooling
Flat turbin
mtmfH;i===;I==== Sterilize
air
Fig. 6.3 Cutaway diagram of a fermenter used for penicillin production.

Many bench-scale fermenters are made of glass with a stainless
steel top plate. As mentioned earlier, larger fermenters are made of
stainless steel (Type 316). The height-to-diameter ratio of the vessels
is either two-to-one or three-to-one. It is usually agitated with two or
three turbine impellers. The impeller shaft enters the vessel either
from the top or the bottom of the vessel through a .bearing housing
and mechanical seal assembly. The impeller diameter to tank
diameter ratio (D//D T ) is generally 0.3 to 0.4. In the case of a two-
impeller system, the distance between the first impeller and the
bottom of the vessel and between the two impellers is 1.5 impeller
diameters. The distance is reduced to one impeller diameter in the
case of a three-impeller system. Four equally spaced baffles are
usually installed to prevent a vortex formation which reduces the
mixing efficiency. The width of the baffle is usually about one tenth of
the tank diameter. For aerobic fermentation, a single orifice sparger
or ring sparger is used to aerate the fermenter. The sparger is located
between the bottom impeller and the bottom of the vessel. The pH in
a fermenter can be maintained by employing either a buffer solution
or a pH controller. The temperature is controlled by heating or

cooling as the system requires.
6.4.1 Batch or Plug-Flow Fermenter

An ideal stirred fermenter is assumed to be well mixed so that the
contents are uniform in composition at all times. Another ideal
fermenter is the plug-flow fermenter, the analysis of which is
analogous to the ideal batch fermenter.
In a tubular-flow fermenter, nutrients and microorganisms enter one
end of a cylindrical tube and the cells grow while they pass through.
Since the long tube and lack of stirring device prevents complete
mixing of the fluid, the properties of the flowing stream will vary in
both longitudinal and radial direction. However, the variation in the
radial direction is small compared to that in the longitudinal
direction. The ideal tubular-flow fermenter without radial variations
is called a plug-flow fermenter (PFF).
In reality, the PFF fermenter is hard to be found. However, the
packed-bed fermenter and multi-staged fermenter can be
approximated as PFF. Even though the steady-state PFF is operated
in a continuous mode, the cell concentration of an ideal batch
fermenter after time t will be the same as that of a steady-state PFF at
the longitudinal location where the residence time i is equal to t
(Figure 6.4). Therefore, the following analysis applies for both the
ideal batch fermenter and steady-state PFF.
If liquid medium is inoculated with a seed culture, the cell will
start to grow exponentially after the lag phase. The change of the cell
concentration in a batch fermenter is equal to the growth rate of cells
in it:
-dC x = r x = J1 Cx (6.20)
dt
To derive the performance equation of a batch fermentation, we
need to integrate Eq. (6.20) to obtain:
J~x dCrxx = tc
Xo
x
Xo
dC x = fdt
J1 C x to
=t - to (6.21)
It should be noted that Eq. (6.21) only applies when rx is larger

than zero. Therefore, t 1 in Eq. (6.21) is not the time that the culture
was inoculated, but the time that the cells start to grow, which is the
beginning of the accelerated growth phase.
According to Eq. (6.21), the batch growth time t - to is the area
under the l/r x versus Cx curve between CXo and Cx as shown in
Figure 6.5. The solid line in Figure 6.5 was calculated with the Monod
equation and the shaded area is equal to t - to. The batch growth time
is rarely estimated by this graphical method since the ex versus t
curve is a more straightforward way to determine it. However, the

graphical representation is useful in comparing the performance of
the various fermenter configurations, which is discussed later. At this
time just note that the curve is U shaped, which is characteristic of
auto catalytic reactions:
Cxo ~
F C ...... !_-+- [~F
- JI"""
CX
C!
So Sj
0+0 to t 'tp
V; Cx Cs Cx
Cs
Cx = Cxo at t = to
Cs = CSo
(a) (b)
Fig. 6.4 Schematic diagram of (a) batch stirred-tank fermenter and

plug-flow fermenter
S+X ~X+X
The rate for an autocatalytic reaction is slow at the start because
the concentration of X is low. It increases as cells multiply and
reaches a maximum rate. As the substrate is depleted and the toxic
products accumulate, the rate decreases to a low value.
If Monod kinetics adequately represents the growth rate during
the exponential period, we can substitute Eq. (6.11) into Eq. (6.21) to
obtain
t x
c Xo
(K s +Cs)dCx = fdt
J1max CS CX to
(6.22)
Eg. (6.22) can be integrated if we know the relationship between Cs

and ex. It has been observed frequently that the amount of cell mass
produced is proportional to the amount of a limiting substrate
consumed. The growth yield (Y XIS) is defined as
YXIS = - - =
~Cx Cx -cx 0 (6.23)
-~Cs -(Cs-C sn )
Substitution of Eq. (6.23) into Eq. (6.22) and integration of the

resultant equation gives a relationship which shows how the cell
concentration changes with respect to time:
- ( K5 YxI 5 1] 1 Cx KsYx/ S 1 CSo
(t - t 0) J.1max- + n + n (6.24)
CXo+CsoYx/s (x, (XI+CSOYX/s Cs
o 2 4 6 8
Fig. 6.5 A graphical representation of the batch growth time t - t1

(shaded area). The solid line represents the Monad model
with ,Jimax = 0.935 hr- 1 , K s =0.71 gIL, Yx/s = 0.6, Gxo = 1
gIL, and Gso = 10 gIL.
The Monod kinetic parameters, Ilmax and K s, cannot be estimated

with a series of batch runs as easily as the Michaelis-Menten
parameters for an enzyme reaction. In the case of an enzyme reaction,
the initial rate of reaction can be measured as a function of substrate
concentration in batch runs. However, in the case of cell cultivation,
the initial rate of reaction in a batch run is always zero due to the
presence of a lag phase, during which Monod kinetics does not apply.
It should be noted that even though the Monod equation has the
same form as the Michaelis-Menten equation, the rate equation is
different. In the Michaelis-Menten equation,
dC p = rmaxC S (6.25)
dt K M +C s
while in the Monod equation,
dC x = J.lmaxCsCX (6.26)
dt Ks + C s
There is a Cx term in the Monod equation vvhich is not present in
the Michaelis-Menten equation.
Example 6.1
The growth rate of E. coli be expressed by Monod kinetics with the
parameters of J.1max = 0.935 hr- and Ks = 0.71 giL (Monod, 1949).
Assume that the cell yield YXIS is 0.6 gdry cells per g substrate. If CXo
is 1 giL and CSt = 10 giL when the cells start to grow exponentially, at
to = 0, show how In Cx, Cx, Cs, d In Cxldt, and dCxl dt change with
respect to time.
Solution:
You can solve Eqs. (6.23) and (6.24) to calculate the change of Cx and
Cs with respect to time, which involves the solution of a nonlinear
equation. Alternatively, the Advanced Continuous Simulation
Language (ACSL, 1975) can be used to solve Eqs. (6.22) and (6.23).
Table 6.1 ACSL Program for Example 6.1
CONSTANT MUMAX .. =0.935, KS=O.71, YXS=O.6,
CX2=1., CS2=10, TSTOP=10
CINTERVAL CINT=O.l $ 'COMMUNICAITON INTERVAL 1
VARIABLE T=O.O
CX=INTEG(MUMAX*CS*CX/(KS+CS) ,CX2)
CS=CS2-(CX-CX2)/YXS
LNCX=ALOG(CX)
DLNCX=MUMAX*CS/(KS+CS)
DCXDT=MUMAX*CS*CX/(KS+CS)
TERMT(CS.LE.O) END
END
In Cx
O~--_...L.--_--~--_......L--
10
Cx
C
5
Cs
O'------...&....----~---......L--
2 3
Fig. 6.6 Solution to Example 6.1.
Table 6.1 shows the ACSL program and Figure 6.6 shows the
results. Comparison of Figure 6.6 with Figure 6.1 shows that Monod
kinetics can predict the cell growth from the start of the exponential
growth phase to the stationary phase.
6.4.2 Ideal Con tin uous Stirred-tank Fermen ter

Microbial populations can be maintained in a state of exponential
growth over a long period of time by using a system of continuous
culture. Figure 6.7 shows the block diagram for a continuous stirred-
tank fermenter (CSTF). The growth chamber is connected to a
reservoir of sterile medium. Once growth has been initiated, fresh

medium is continuously supplied from the reservoir.
F CXi
Csi
F Cx
Cs
Fig. 6.7 Schematic diagram of continuous stirred-tank fermenter (CSTF)
Continuous culture systems can be operated as chemostat or as

turbidostat. In a chemostat the flow rate is set at a particular value and
the.rate of growth of the culture adjusts to this flow rate. In a
turbidostat the turbidity is set at a constant level by adjusting the
flow rate. It is easier to operate chemostat than turbidostat, because
the former can be done by setting the pump at a constant flow rate,
whereas the latter requires an optical sensing device and a controller.
However, the turbidostat is recommended when continuous
fermentation needs to be carried out at high dilution rates near the
washout point, since it can prevent washout by regulating the flow
rate in case the cell loss through the output stream exceeds the cell
growth in the fermenter.
The material balance for the microorganisms in a CSTF (Figure 6.7)
can be written as
dC
x
FC x ' - FC x + Vr x = V-- (6.27)
I dt
where r x is the rate of cell growth in the fermenter and dCx/dt
represents the change of cell concentration in the fermenter with
time.
For the steady-state operation of a CSTF, the change of cell
concentration with time is equal to zero (dCx/dt = 0) since the
microorganisms in the vessel grow just fast enough to replace those
lost through the outlet stream, and Eq. (6.27) becomes
V Cx -Cx.
T.
m-
- _
F -
- I
(6.28)
rx
Eq. (6.28) shows that the required residence time is equal to
Cx -C Xi ' times l/rx, which is equal to the area of the rectangle of
width Cx - CXi and height l/rx on the l/rx versus Cx curve.
Figure 6.8 shows the l/rx versus Cx curve. The shaded rectangular
area in the figure is equal to the residence time in a CSTF when the
inlet stream is sterile. This graphical illustration of the residence time
can aid us in comparing the effectiveness of fermenter systems. The
shorter the residence time in reaching a certain cell concentration, the
more effective the fermenter. The optimum operation of fermenters
based on this graphical illustration is discussed in the next section.
If the input stream is sterile (C xI" = 0), and the cells in a CSTF are
growing exponentially '(rx = J.1C x ), Eq. (6.28) becomes
1 1
Tm = f.l = D (6.29)
where D is known as dilution rate and is equal to the reciprocal of

the residence time (Tm). Therefore, for the steady-state CSTF with
sterile feed, the specific growth rate is equal to the dilution rate. In
other words, the specific growth rate of a microorganism can be
controlled by changing the medium flow rate. If the growth rate can
be expressed by Monod equation, then
D = f.l = ~ = f.lmaxCs (6.30)

Tm K s + Cs
From Eq. (6.30), Cs can be calculated with a known residence time

and the Monod kinetic parameters as:
Cs = Ks (6.31)
T mJ1max -1
4
o 2 4 6
ex
Fig. 6.8 A graphical representation of the estimation of residence
time for the CSTF. The line represents the Monad model
with J1max = 0.935 hr- 1 , K s = 0.71 gIL, Yx/s = 0.6, CsI.=10
gIL, and CXi ' = o.
It should be noted, however, that the preceding expression is only

valid when Tm J.1max > 1. If Tm J.1max < I, the growth rate of the cells is
less than the rate of cells leaving with the outlet stream.
Consequently, all of the cells in the fermenter will be washed out and
Eq. (6.31) is invalid.
If the growth yield (Y XIS) is constant, then
Cx = YXIS (C Si - Cs) (6.32)
Substituting Eq. (6.31) into Eq. (6.32) yields the correlation for Cx
as
Cx = YXIS (CSi - K
'f mJ.1max
s
-1
) (6.33)
Cp = CPi + YPIS (C Si - Ks ) (6.34)

'f mJ.1max -1
Again, Eqs. (6.33) and (6.34) are valid only when 'fm J.1max >1
In this section, we set a material balance for the microbial
concentration and derive various equations for the CSTF. The same
equations can be also obtained by setting material balances for the
substrate concentration and product concentration.
6.4.3. Evaluation of Monod Kinetic Parameters

The equality of the specific growth rate and the dilution rate of the
steady-state CSTF shown in Eq. (6.30) is helpful in studying the
effects of various components of the medium on the specific growth
rate. By measuring the steady-state substrate concentration at
various flow rates, various kinetic models can be tested and the value
of the kinetic parameters can be estimated. By rearranging Eq. (6.30),
a linear relationship can be obtained as follows:
~ = ~~+_1_ (6.35)
J.1 J.1 max CS J.1. max
where J.1 is equal to the dilution rate (D) for a chemostat. If a certain
microorganism follows Monod kinetics, the plot of 1/ J.1 versus l/C s
yields the values of J.1max and Ks by reading the intercept and the slope
of the straight line. This plot is the same as the Lineweaver-Burk plot
for Michaelis-Menten kinetics. It has the advantage that it shows the
relationship between the independent variable (C s) and dependent
variable (J.1). However, 1/ J.1 approaches infin~ty as the substrate
concentration decreases. This gives undue weight to measurements
made at low substrate concentrations and insufficient weight to
measurements at high substrate concentrations.
Eq. (6.30) can be rearranged to give the following linear
relationships, which can be employed instead of Eq. (6.35) for a better
estimation of the parameters in certain cases.
C Ks C
-s= - - + -s- (636)
)1 )1max )1max
J1 = Jlmax - K s CJ1 (6.37)

s
However, the limitation of this approach to determine the kinetic
parameters is in the difficulty of running a CSTF. For batch runs, we
can even use shaker flasks to make multiple runs with many different
conditions at the same time. The batch run in a stirred fermenter is
not difficult to carry out, either. Since there is no input and output
connections except the air supply and the length of a run is short, the
danger of contamination of the fermenter is not serious.
For CSTF runs, we need to have nutrient and product reservoirs
which are connected to the fermenter aseptically. The rate of input
and output stream needs to be controlled precisely. Sometimes, the
control of the outlet flowrate can be difficult due to the foaming or
plugging by large cell aggregates. Since the length of the run should
last several days or even weeks to reach a steady state and also to
vary the dilution rates, there is always a high risk for the fermenter to
be contaminated. Frequently, it is difficult to reach a steady state
because of the cell's mutation and adaptation to new environment.
Furthermore, since most large-scale fermentations are carried out
in batch mode, the kinetic parameters determined by the chemostat
study should be able to predict the growth in a batch fermenter.
However, due to the significantly different environments of batch
and continuous fermenters, the kinetic model developed from the
CSTF runs may fail to predict the growth behavior of a batch
fermenter. Nevertheless, the verification of a kinetic model and the
evaluation of kinetic parameters by running chemostat is the most
reliable method because of its constant medium environment.
The data from batch runs can be used to determine the kinetic
parameters, even though this is not a highly recommended
procedure. The specific growth rate during a batch run can be
estimated by measuring the slope of the cell concentration versus
time curve at the various points. The substrate concentration needs to
be measured at the same points where the slope is read. Then, the
plots according to Eqs. (6.35), (6.36), (6.37) can be constructed to
determine the kinetic parameters. However, the parameter values
obtained in this method need to be checked carefully whether they
are in the reasonable range for the cells tested.
Example 6.2
A chemostat study was performed with yeast. The medium flow rate
was varied and the steady-state concentration of cells and glucose in
the fermenter were measured and recorded. The inlet concentration
of glucose was set at 100 giL. The volume of the fermenter contents
was 500 mL. The inlet stream was sterile.
Flow rate Cell Cone. Substrate Cone.
F, mL/hr CX, gIL CS , gIL
31 5.97 0.5
50 5.94 1.0
71 5.88 2.0
91 5.76 4.0
200 0 100
a. Find the rate equation for cell growth.

b. What should be the range of the flow rate to prevent washout of
the cells?
Solution:
a. Let's assume that the growth rate can be expressed by Monod
kinetics. If this assumption is reasonable, the plot of 1/ J1
versus 1 /C s will result in a straight line according to Eq. (6.35).
The dilution rate for the chemostat is
D=£
20 V
15
1
D 10
o 0.5 1.0 1.5 2.0

1
Cs
Fig.6.9 The plot of 1/D versus 1/Cs for Example 6.2.
The plot of 1/0 versus l/C s is shown in Figure 6.9 which shows a
straight line with intercept
_1_ =3.8
Jimax
and slope
~=5.2
J.1max
Therefore, J.1max = 0.26 hr-l, and K s = 1.37 giL. The rate equation
of cell growth is
0.26C S Cx
rx =
1.37 + Cs
b. To prevent washout of the cells, the cell concentration should be
maintained so that it will be greater than zero. Therefore, from
Eq. (6.33).
Cx = YX/S(C Si - K
s ) >0
T mJ.1max -1
Solving the preceding equation for Tm yields
V s K +Cs.
r - - - I
m - F - C J.1
Si max
Therefore,
VC s,J.1max = 0.5(100)(0.26) = 1.128 L/hr
F= I
K s +Cs. I
1.37 + 100
6.4.4 Productivity of CSTF

Normally, the productivity of the fermenter is expressed as the
amount of a product produced per unit time and volume. If the inlet
stream is sterile (C Xi = 0), the productivity of cell mass is equal to
CX/Tm' which is equal to the slope of the straight line OAB of the Cx
vs. Tm curve, as shown in Figure 6.10. The productivity at point A is
equal to that at point B. At point A, the cell concentration of the outlet
stream is low but the residence time is short, therefore, more medium
can pass through. On the other hand, at point B the cell concentration
of the outlet stream is high, but the residence time is long, so a
smaller a.mount of medium passes through. Point A is an unstable
region because it is very close to the washout point D, and because a
small fluctuation in the residence time can bring about a large change
in the cell concentration. As the slope -.2!. the line increases, the
productivity increases, and the length of AB decreases. The slope of
the line will have its maximum value when it is tangent to the Cx
curve. Therefore, the value of the maximum productivity is equal to
the slope of line OC . The maximum productivity will be attained at
the point D.
The operating condition for the maximum productivity of the

CSTF can be estimated graphically by using 1/ rx versus Cx curve.
The maximum productivity can be attained when the residence time
is the minimum. Since the residence time is equal to the area of the
rectangle of width Cx and height 1/ rx on the 1/ rx versus Cx curve, it
is the minimum when the 1/r x is the minimum, as shown in Figure
6.11.
10
Cx 6
C B
4
2 A
4 6
Fig. 6.10 The change of the concentrations of cells and substrate

as a function of the residence time. Productivity is equal
to the slope of the straight line OAB . The curve is drawn
1
by using the Monad model with ,J1max = 0.935 hr- , Ks =
0.71 gIL, Yx/s = 0.6, and Cs;= 10 gIL.
4
3
1
rx 2
o 2 4 6
Cx
Fig. 6.11 A graphical illustration of the CSTF with maximum
productivity. The solid line represents the Monad model
= =
with J1max = 0.935 hr-1, Ks 0.71 gIL, YXIS 0.6,
Cs; = 10 gIL, and Cx ; =o.
It would be interesting to derive the equations for the cell
concentration and residence time at this maximum cell productivity.
The cell productivity for a steady-state CSTF with sterile feed is
C x = r = IlmaxCsCX (6.38)
x
t'm K s +C s
The productivity is maximum when drxldC x = o. After substituting

Cs = C Si - Cx Y XIS into the preceding equation, differentiating with
respect to Cx, and setting the resultant equation to zero, we obtain the
optimum cell concentration for the maximum productivity as
Cx,opt = YX/SC Si ~1
a+
(6.39)
where
a= ~Ks +CSi (6.40)

Ks
Since Cs - CsilY xiS
C
C _ _S_i ( )
S.opt - a +1 6.41
Substituting Eg. (6.41) into Eq. (6.38) for Cs yields the optimum
residence time:
a
(6.42)
Tm,opt =
J1 m ax (a-
1)
6.4.5 Comparison of Batch and CSTF

As discussed earlier, the residence time required for a batch or
steady-state PPP to reach a certain level of cell concentration is
(6-43)
where to is the time required to reach an exponential growth phase.

The area under the 11rx versus Cx curve between Cx and Cx is equal
to tb - tlo, as shown in Figure 6.5.
On the other hand, the residence time for the CSTF is expressed as
Eq. (6.28) which is equal to the area of the rectangle of width Cx - CXi
and height llrx.
Since the 11r x versus Cx curve is U shaped, we can make the
following conclusions for single fermenter.
1. The most productive fermenter system is a CSTF operated at
the cell concentration at which value of 11rx is minimum, as
shown in Figure 6.12(a), because it requires the smallest
residence time.
2. If the final cell concentration to be reached is in the stationary
phase, the batch fermenter is a better choice than the CSTF
because the residence time required for the batch as shown in
Figure 6.12(b) is smaller tllan that for the CSTF.
1 1
'x 'x
Fig. 6.12 Graphical illustration of the residence time required

(shaded area) for the (a) CSTF and (b) Batch fermenter.
1 1
rx rx
Fig. 6.13 Graphical illustration of the total residence time required

(shaded area) when two fermenters are connected in
series: (a) CSTF and PFF, and (b) two CSTFs.
6.5 MULTIPLE FERMENTERS CONNECTED IN SERIES

A question arises frequently whether it may be more efficient to use
multiple fermenters connected in series instead of one large
fermenter. Choosing the optimum fermenter system for maximum
productivity depends on the shape of the l/r x versus C x curve and
the process requirement, such as the final conversion.
In the 1/ r x versus Cx curve, if the final cell concentration is less
than Cx,opt one fermenter is better than two fermenters connected in
series, because two CSTFs connected in series require more residence
time than one CSTF does. However, if the final cell concentration is
much larger than Cx,opt the best combination of two fermenters for a
minimum total residence time is a CSTF operated at Cx,opt followed
by a PFF, as shown in (Figure 6.13a). A CSTF operated at Cx,opt
followed by another CSTF connected in series is also better than one
CSTF (Figure 6.13b).
6.5.1 CSTF and PFF in Series

Figure 6.14 shows the schematic diagram of the two fermenters
connected in series, CSTF followed bv PFF. The result of the material
balance for the first fermenter is the same as the single CSTF which
we have already developed. If the input stream is sterile (C Xi = 0), the
concentrations of the substrate, cell and product can be calculated
from Eqs. (6.31),6.33), and 6.34), as follows:
F Cx;
Cs;
F Cx2
Cs2
Fig. 6.14 Schematic diagram of the two fermenters,

CSTF and PFF, connected in series.
Ks
C51 = (6.44)
r m1 /lmax -1
CX1=YX/S(CSi-r flK
111 1
S
max
-1) (6.45)
CPt = CPi + Y p/ s (CSi - K

s ) (6.46)
'111 1 /lmax -1
For the second PFF, the residence time can be estimated by

r =
P2
t
CX1
X2 dCx
rx
= t
CX1
X2 (K s + Cs)dCx
/lmaxCsCX
(6.47)
Since growth yield can be expressed as

C -Cx
y -_ x2 1 (6.48)
XIS - C C
51 - 52
Integration of Eq. (6.47) after the substitution of Eq. (6.48) will
result,
_( KsYx/s ) CX2 KsYx / s CS1 (6.49)

'p2/lmax - + 1 In- + In-
ex L + Cs1 Yx / s Cx1 Cx1 + Cs1 Y x / s Cs2
.}f the final cell concentration (C X2 ) is known, the final
substrate concentration (C sz ) can be calculated from Eq. (6.48). The
residence time ofthe second fermenter can then be calculated using
Eq. (6.49). If the residence time of the second fermenter is known,
Eqs. (6.48) and (6.49) have to be so~ved simultaneously to estimate

the cell and substrate concentrations. Another approach is to
integrate Eq. (6.47) numerically until the given t p2 value is reached.
6.5.2 Multiple CS TFs in Series

If the final cell concentration is larger than Cx,opt, the best
combination of two fermenters for a minimum total residence time is
a CSTF operated at Cx,opt followed by a PFF, as explained already.
However, the cultivation of microorganisms in the PFF is limited to
several experimental cases, such as the tubular loop batch fermenter
(Russell et al., 1974) and scraped tubular fermenter (Moo-Young et
al., 1979). Furthermore, the growth kinetics in a PFF can be
significantly different from that in a CSTF.
Another more practical approach is to use multiple CSTFs in
series, since a CSTF operated at Cx,opt followed another CSTF
connected in series is also better than one CSTF.
Hill and Robinson (1989) derived an equation to predict the
minimum possible total residence time to achieve any desired
substrate conversion. They found that three optimally designed
CSTFs connected in series provided close to the minimum possible
residence time for any desired substrate conversion.
Figure 6.15 shows the schematic diagram of the multiple CSTFs
connected in series. For the nth steady-state CSTF, the material
balance for the microorganisms can be written as
F(C Xn _1 - Cxn ) + VnR xn =0 (6.50)
CX1 Cxn- 1
CS1 ~--f--------. r-···C~n-1 r--~-+-----.
Fig. 6.15 Schematic diagram of the multiple CSTFs connected in series.
J1maxCSnCX
rx = n (6.51)
n K s + CSn
Growth yield can be expressed as
y _ C x -Cx
(6.52)
II n-·(
xIS - C C
SIl-l - S/I
By solving Eqs. (6.50), (6.51), and (6.52) simultaneously, we can

calculate either dilution rate with the known cell concentration, or
vice versa.
The estimation of the cell or substrate concentration with the
known dilution rate can be done easily by using graphical technique
as shown in Figure 6.1 (Luedeking, 1967). From Eq. (6.50), the
dilution rate of the first reactor when the inlet stream is sterile is
F rx
01 = - = _ 1 (6.53)
VI ex1
which can be represented by the slope of the straight line
connecting the origin and (CXiS X ) in Figure 6.16. Similarly, for the
second ferm.enter
F rX
°2= - 2
= ---- (6.54)
Cx2 -CXl
V2
which is the slope of the line connecting (C X1 ,= 0) and (C x2 ,rX2 ).
Therefore, by knowing the dilution rate of each fermenter, you can
estimate the cell concentration of each fermenter, or vice versa.
Example 6.3
Suppose you have a microorganism that obeys the Monad
equation:
dC x = /lmaxCsCX
dt K s + Cs
where /ln1Clx = 0.7 hr- 1 and K s = 5 g/I.J. The cell yield (Y XIS) is 0.65.
You want to cultivate this microorganism in either one fermenter or
two in series. The flow rate and the substrate concentration of the
inlet stream should be 500 L/hr and 85 g/l.J, respectively. The
substrate concentration of the outlet stream must be 5 giL
4
01L.-._~_--1..--_---L-.-_-..L....L.-.---L_....J.-..--,I
o 234 5 6
Cx
Fig. 6.16 Graphical solution for a two-stage continuous fermentation.
The line represents the Monod model with ,J.1max =0.935 hr- ,
1
Ks =0.71 gIL, Yx/s=0.6, CSi =10g/L,Cxi = O.}

a. If you use one CSTF, what should be the size of the fermenter?
What is the cell concentration of the outlet stream?
b. If you use two CSTFs in series, what sizes of the two fermenters
will be most productive? What are the concentration of cells
and substrate in the outlet stream of the first fermenter?
c. What is the best combination of fermenter types and volumes if
you use two fermenters in series?
Solution:
a. For a single steady-state CSTF with a sterile feed, the dilution
rate is equal to specific growth rate:
D =£ = Ilmax Cs = 0.7(5) = 0.35

V K s + Cs 5+ 5
V =£ = 500 = 1 429 L
D 0.35 '
The cell concentration of the outlet stream is
C x = Yx/s(C Si - Cs) = 0.65 (85 - 5) = 52 giL
b. For two CSTFs in series, the first fermenter must be operated at
Cx,opt and Cs,opt
F CXi Cx1
CSi Cs1~__~__~
Therefore, from Eq. (6.39) through Eq. (6.42),
a= ~KS+CSi = ~5+85 =4.2

Ks 5
e Xl = Cx
opt
= Yx/s Cs '
1
~
a +1
= O.65(85)~ = 45g/L
4.2 + 1
Cs. 85
Cs = Cs = __ = __ = 1.6 giL
1
1 opt a +1 4.2 + 1
a 4.2
! 1'111 = !mopt -- = 1.9 hr
,umax(a -1) 0.7(4.2 -1)
VI rmIF = 1.9(500) = 950 L

For the second fermenter, from Eq. (6.50),
F(C - C )+
v:2,-,max C Cx
II
S2 2 =0
Xl X2 K +C
S S2
By rearranging the preceding equation for V 2,
F(C x - Cx ) = 500(52 - 45) = 192 L

-
V 2- 2 1
/lmaxCs2 Cx 2 /(K s + Cs2 ) 0.7(5)(52)/(5 + 5)
The total volume of the two CSTFs is

V = VI + V 2 = 950 + 192 = 1,142 L
which is 20 percent smaller than the volume required when we
use a single CSTF.
c. The best combination is a CSTF operated at the maximum rate
followed by a PFF. The volume of the first fermenter is 950 L as
calculated in part (b).
For the second PFP, from Eq. (6.54)
r, = -1- (KsYx / s X2
+lln-+
) C In-
KsYx / s CS1
P2 J.1max + C]
Cx1 s Yx/s Cx1 Cx1 + Cs1 + Cs1 Yx / s Cs2
=~[( 5(0.65) +1)ln~3+ 5(0.65) In 16 ]=0.32

0.7 45 + 16(0.65) 45 45 + 16(0.65) 5
Therefore,
V 2 r p2 F = 0.32 (500) = 160L
The total volume of the CSTF and PFF is
V = VI + V 2 = 950 + 160 = 1,110 L

which is 22 percent smaller than the volume required when we use
a single CSTF. The additional saving by employing the second PFF
instead of a second CSTF is not significant in this case.
6.6 CELL RECYCLING

For the contiIluoUS operation of a PFF or CSTF, cells are discharged
with the outlet stream which limits the productivity of fermenters.
The productivity can be improved by recycling the cells from the
outlet stream to the fermenter.
F Cxi "r--.--------. B Cxf

Csi CSf
RF
CxR,Cst L CxL =0
Cst
Fig.6.17 Schematic diagram of PFF with cell recycling.
6.6.1 PFF with Cell Recycling

A PFF requires the initial presence of microorganisms in the inlet
stream as a batch fermenter requires initial inoculum. The most
economical way to provide cells in the inlet stream is to recycle the
part of the outlet stream back to the inlet with or without a cell
separation device. Figure 6.17 shows the schematic diagram of a PFF
with cell recycling. Unlike the CSTF, the PFF does not require the cell
separator in order to recycle, though its presence increases the
productivity of the fermenter slightly as will be shown later. The
performance equation of the PFF with Monod kintics can be written
as:
v = --.!..E..- = reXf dC x = tXf (K s + Cs )dC x (6.55)
(1 + R)F 1+ R Jc x rx Cx J1maxCsCX
where Tp is the residence time based on the flow rate of the overal
system. The actual residence time in the fermenter is larger than Tp
due to the increase of the flow rate by the recycling.
If the growth yield is constant,
Cs = C s- _1_
Yx / s
(C x - Cx) (6.56)
Substituting Eq. (6.56) into Eq. (6.55) for Cs and integrating it will
result:
T pf.1max
--- =
(KsYx / s ) CXf
+lln-, + ,
KsYx / s C~
In- (6.57)
l+R Cx+CsYx / s Cx Cx+CsYx / s CSf
where C~ and Cs can be estimated from the cell and substrate
balance at the mixing point of the inlet and the recycle stream as
, Cx +RCx
C - (6.58)
x-
i . R
l+R
(6.59)
The cell concentrations of the outlet stream, can be estimated from

the overall cell balance
1
CX! = 13 [Cx; + Yx/s(Cs; - CSt)] (6.60)
The cell concentrations of the recycle stream, can be estimated
from the cell balance over the filter as
C - l+R+fJ (6.61)
XR - R Cx
f
where f3 is the bleeding rate which is defined as

13= B (6.62)
F
Figure 6.18 shows the effect of the recycle rate (R) on the residence
time of a PFF system with recycling. Note that r is calculated based
on the inlet flowrate that is the true residence time for the fermenter
system. The actual r in the PFF is unimportant because it decreases
with the increase of the recycle rate. When f3 =1, the bleeding rate is
equal to the flow rate and the flow rate of filtrate stream L is zero,
therefore, the recycle stream is not filtered. The residence time will be
infinite if R is zero and decreases sharply as R is increased until it
reaches the point the decrease is gradual. In this specific case, the
optimum recycle ratio may be somewhere around 0.2.
8---y--------------------,
V
't
P-
- -
F 4
~ = 0.8
O-+---.,.---r---r---r---r----r---....,-----r---,..-------t
0.0 0.2 0.4 0.6 0.8 1.0
Fig. 6.18 The effect of the recycle rate (R) and the bleeding rate
(f3 = B/F) on the residence time (r = V/F hr) of a PFF system
with recycling. The curve is drawn by using the Monad model
= = =
with, J1max 0.935 hr-\ K s 0.71 gIL, Yx/s 0.6, Cs; =10 gIL,
CSf' = 1.3 gIL, and ex;, = o.
Another curve in Figure 6.18 is for f3 = B/F = 1.8. The required

residence time can be reduced by concentrating the recycle stream 25
to 40% when R is between 0.2 and 1.0. When R ~ 1.2, the part of curve
is noted as a dotted line because it may be difficult to reduce the
recycle ratio below 0.2 when f3 = 0.8. For example, in order to
maintain R = 0.1, The amount of 1.3F needs to be recycled and
concentrated to O.lF, which may be difficult depending on the cell
concentration of the outlet. The higher the concentration factor in a
filter unit is, the higher the danger of the filter failure can be
expected.
The analysis in this section and the next can be also applied for a
cell settler as a cell separator. The outlet stream of the cell settler will
be equal to F = B + L and its concentration will be (B / F)C xf = f3Csj"
F Cx;
Cs ;
L CxL = 0
Cs
.·:::::v:::···
Fig.6.19 Schematic diagram of CSTF with cell recycling.
6.6.2 CSTF with Cell Recycling

The cellular productivity in a CSTF increases with an increase in the
dilution rate and reaches a maximum value. If the dilution rate is
increased beyond the maximum point, the productivity will be
decreased abruptly and the cells will start to be washed out because
the rate of cell generation is less than that of cell loss from the outlet
stream. Therefore, the productivity of the fermenter is limited due to
the loss of cells with the outlet stream. One way to improve the
reactor productivity is to recycle the cell by separating the cells from
the product stream using a cross-flow filter unit (Figure 6.19).
The high cell concentration maintained using cell recycling will
increase the cellular productivity since the growth rate is
proportional to the cell concentration. However, there must be a limit
in the increase of the cellular productivity with increased cell
concentration because in a high cell concentration environment, the
nutrient-transfer rate will be decreased due to overcrowding and
aggregation of cells. The maintenance of the extremely high cell
concentration is also not practical because the filter unit will fail more
frequently at the higher cell concentrations.
If all cells are recycled back into the fermenter, the cell
concentration will increase continuously with time alld a steady state
will never be reached. Therefore, to operate a CSTF with recycling in
a steady-state mode, we need to have a bleeding stream, as shown in
Figure 6.19. The material balance for cells in the fermenter with a cell
recycling unit is
dC x
FC x · - BC x + V J.1 Cx = V - (6.63)
1 dt
It should be noted that actual flow rates of the streams going in
and out of the filter unit do not matter as far as overall material
balance is concerned. For a steady-state CSTF with cell recycling and
a sterile feed,
10
- - ~=0.5
8
................. No Recycle
DCx 6
2
\
.................•
o
0.0 0.5 1.0 1.5 2.0
o
Fig. 6.20 The effect of bleeding ratio on the cellular productivity
(f3DC x )·
f3D = ~ = J.l (6.64)

rm
Now, 13 D instead of D is equal to the specific growth rate. When
{3 =1, cells are not recycled, therefore, D = Ji.
If the growth rate can be expressed by Monod kinetics,
substitution of Eq. (6.11) into Eq. (6.64) and rearrangement for Cs
yields
-
Cs- 13 K s (6.65)
r mil max - f3
which is valid when 'fm J.1max > 13.' The cell concentration in the
fermenter can be calculated from the value of Cs as
(6.66)
Figure 6.20 shows the effect of bleeding ratio on the cell

productivity for the Monod model. As f3 is reduced from 1 (no
recycling) to 0.5, the cell productivity is doubled.
6.7 ALTERNATIVE FERMENTERS

Many alternative fermenters have been proposed and tested. These
fermenters were designed to improve either the disadvantages of the
stirred tank fermenter-high power consumption and shear damage,
or to meet a specific requirement of a certain fermentation process,
such as better aeration, effective heat removal, cell separation or
retention, immobilization of cells, the reduction of equipment and
operating costs for inexpensive bulk products, and unusually large
designs.
Fermenters are usually classified based on their vessel type such as
tank, column, or loop fermenters. The tank and column fermenters
are both constructed as cylindrical vessels. They can be distinguished
based on their height-to-diameter ratio (HID) as (Schiigerl, 1982):
HID < 3 for the tank and
HID> 3 for the column fermenter
A loop fermenter is a tank or column fermenter with a liquid
circulation loop, which can be a central draft tube or an external loop.
Table 6.2 Classifications of Fermenters
Primary Source of Mixing

Vessel Compressed Internal External
Type Air Moving Parts Pumping
Tank - stirred-tank -
Column bubble column multistage sieve tray
tapered column (or cascade) packed-bed
Loop air-lift propeller loop jet loop
pressure cycle
Another way to classify fermenters is based on how the fermenter

contents are mixed: by compressed air, by a mechanical internal moving
part, or by external liquid pumping. Representative fermenters in
each category are listed in Table 6.2 and the advantages and
disadvantages of three basic fermenter types are listed in Table 6.3.
6.7.1 Column Fermenter

The most simple fermenter is the bubble column fermenter (or tower
fermenter), which is usually composed of a long cylindrical vessel
with a sparging device at the bottom [Figure 6.21 (a)-(c)]. The

fermenter contents are mixed by the rising bubbles which also
provide the oxygen needs of the cells. Since it does not have any
moving parts, it is energy efficient with respect to the amount of
oxygen transfer per unit energy input. As the cells settle, high cell
concentrations can be maintained in the lower portion of the column
without any separation device.
Table 6.3 Advantages and Disadvantages of Three Basic Fermenter
Configurations
Type Advantages Disadvantages
1. Flexible and adaptable 1. High power consumption
2. Wide range of mixing 2. Damage shear sensitive
Stirred- intensity cells
tank 3. Can handle high viscosity 3. High equipment costs
media
1. No moving parts 1. Poor mixing
Bubble 2. Simple 2. Excessive foaming.
Column 3. Low equipment costs 3. Limited to low viscosity system
4. High cell concentration
1. No moving parts 1. Poor mixing
Air-lift 2. Simple 2. Excessive foaming
3. High gas absorption 3. Limited to low viscosity system
efficiency
4. Good heat transfer
However, the bubble column fermenter is usually limited to

aerobic fermentations and the rising bubbles may not provide
adequate mixing for optimal growth. Only the lower part of the
column can be maintained with high cell concentrations, which leads
to the rapid initial fermentation followed by a slower one involving
less desirable substrates. As the cell concentration increases in a
fermenter, high air-flow rates are required to maintain the cell
suspension and mixing. However, the increased air-flow rate can
cause excessive foaming and high retention of air bubbles in the
column which decreases the productivity of the fermenter. As
bubbles rise in the column, they can coalesce rapidly leading to a
decrease in the oxygen-transfer rate. Therefore, column fermenters
are inflexible and limited to a relatively narrow range of operating
conditions.
To overcome the weaknesses of the column fermenter, several
alternative designs have been proposed. A tapered column fermenter
[Figure 6.21 (b)] can maintain a high air-flow rate per unit area at the
lo"ver section of the fermenter where the cell concentration is high.
Cell Kinetics and Fernlenter Design 161
Several sieve plates can be installed in the column [Figure 6.21 (c)] for
the effective gas-liquid contact and the breakup of the coalesced
bubbles. To enhance the mixing witholtt internal moving parts, the
fermentation broth can be pumped out and recirculated by using an
external liquid pump [Figure 6.21 (d)(e)].
Air
(a) (b) (c) (d) (e)
Fig. 6.21 Column fermenters: (a) bubble column, (b) tapered bubble
column, (c) sieve-tray bubble column, (d) sieve-tray column
with external pumping, and (e) packed-bed column with
external pumping.
6.7.2 Loop Fermenter

A loop fermenter is a tank or column fermenter with a liquid
circulation loop, which can be a central draft tube or external loop.
Depending on how the liquid circulation is induced, it can be
classified into three different types: air-lift, stirred loop, and jet loop
(Figure 6.22).
The liquid circulation of the air-lift fermenter is induced by
sparged air which creates a density difference between the bubble-
rich part of the liquid in the riser and the denser bubble-depleted part
of the liquid in the downcomer as shown in Figure 6.22 (a). The liquid
circulation and mixing can be enhanced by by circulating liquid
externally using a pump as shown in Figure 6.22 (b). However,
adding the pump diminishes the real advantages of an air-lift
fermenter for being simple and energy efficient.
The leI pressure cycle fermenter (Imperial Chemical Industries
Ltd., England) is an air-lift fermenter with an outer loop, which was
developed for the aerobic fermentation requiring heat removal such
as the single-cell protein production from methanol. Medium and air
are introduced into the upper and lower parts of the loop as shown in
Figure 6.22 (c). The air serves two purposes: It provides the oxygen
needed for the growth of the microorganisms and the rising air
creates natural circulation of the liquid in the fermenter through the
loop. A heat exchanger to cool the liquid medium is installed in the
loop. It was claimed that the fermenter gives a high rate of oxygen
absorption per unit of volume, that it uses a high proportion of
oxygen in the air passed through the fermenter, and that the high
circulation of the fermentation liquor provides good mixing
(Technical Brochure, leI Ltd.).
6.8 STRUCTURED MODEL

So far, the kinetic models described in this chapter have been
unstructured, distributed models based on assumptions that cells can
be represented by a single component, such as cell mass or cell
number per unit volume and the population of the cellular mass is
distributed uniformly throughout the culture. These models do not
recognize the change in the composition of cells during growth.
Unstructured, distributed models such as Monod's equation can
satisfactorily predict growth beha\Tior of many situations. However,
they cannot account for lag phases, sequential uptake of substrates,
or changes in mean cell size during the growth cycle of batch culture.
Structured models recognize the multiplicity of cell components
and their interactions. Many different models have been proposed
based on the assumptions made for cell components and their
interactions. More comprehensive review of these models can be
found in Tsuchiya et al. (1966) and Harder and Roels (1982). In this
section, general equations describing structured models are derived
and a simple structured model is introduced.
6.8.1 General Structured Model

Let's define a system as an individual cell or multiple cells. The
system does not contain any of the abiotic phase of culture. Instead, it
is the biotic2 phase only, which possesses mass m Ofl a dry basis and
specific volume v. Let's assume that there are c components in the
ct;..ll and the mass of the jth component per unit volume of system is
(ex.) . It is also assumed that there exist kinetic rate expressions for p
rea~tions occurring in the system and the rate of jth component
r
formed from the ith reaction per unit volume of system is x 1,1..
Then, during batch cultivation, the change of the jth component in
the system with respect to time can be expressed as (Fredrickson,
1976):
d(mvC\f) A P
= mv Lrx. (6.67)
dt i= 1 I.'
If we assume that the specific volume v is constant with time,
Eq. (6.67) can be rearranged to
2 Caused or produced by living beings.

t
~ ~ i
Air
(a) (b) (c)
Fig. 6.22 Loop fermenters: (a) air-lift, (b) air-lift with external
pumping (c) lei pressure cycle.
dC-x 1 dm
~rx··
-dt-j -- i=l
£..J
p
---c
dt x·
A
(6.68)
I,} m J
where the second term of the right-hand side of Eq. (6.68) represents
the dilution of intracellular components by the growth of
biomaterial. It should be noted that all variables denoted by
circumflexes in the preceding equations are intracelluLar properties.
Since the structural models recognize the multiplicity of cell
components and their interactions in the cell, it makes more sense to
express the model with intrinsic variables.
Since Cx :::: 'L;=1 Cx j the summation of Eq. (6.68) for all c compo-
nents yields
P
A
dCx _
-- -
f ~ A
£..J LJrx.. -
1 dm C"
x (6.69)
dt j = 1 i =1 1,1 m dt
where ex is the mass of biomaterial per unit volume of the biotic
system that is equal to 1/ v,
which was assumed to be constant.
Therefore, Eq. (6.69) is simplified to
m dt
~dm = ~±
ex j=l i=l I,J
IJx (6.70)
which gives the relationship between the rate of cell growth and the
kinetic rate expressions of all reactions occurring in cells.
The concentration terms m the preceding equations can be
expressed as mass per unit culture volume V instead of that per biotic
system volume m v. The two different definitions of concentration are
related as
164 Fundamentals of Biochenzical Engineering
v
cx. J
= -ACX.
mv J
(6.71)
Note that the concentration based on the culture volume is no

longer denoted with a circumflex. Substituting Eq. (6.71) into Eq.
(6.68) and simplifying for the constant V yields
dC xj mv P
----at = V oLrXioi
A
(6.72)
1=1
It should be noted that even though concentration terms are
expressed based on the total culture volume, kinetic parameters still
remain on a biotic phase basis in the formulation.
6.8.2 Two-Compartment Model

One of the simplest structured models is the two-compartment
model proposed by Williams (1967) and is based on the following
assumptions: 3
1. The cell comprises two basic compartments: a synthetic portion
A such as precursor molecules and RNA, and a structural
portion B such as protein and DNA.
,., A "-
ex = CxA +Cx B (6.73)

where ( ex is equal to 1 v, and assumed to be constant,
dC x = 0 (6.74)
dt
2. The synthetic portion A is fed by uptake from a substrate Sand
the structural portion B is in turn fed from the synthetic portion
as:
S-~A-~B (6.75)
3. The rate of the first reaction in Eq. (6.75) is proportional to the
product of substrate and cell concentrations as:
rx I,A
C
= k1CS X (6.76)
where Cs is the mass of substrate per unit abiotic volume, V-m v. The
rate of the second reaction in Eq. (6.75) is proportional to the product
of the concentrations of the synthetic portion and the structural
portion as:
(6.77)
3 The original Williams model (1967) was modified according to the suggestion made by
Fredrickson (1976).
If we write Eq. (6.68) for each component, and substitute Eqs.

(6.76) and (6. 77) for the reaction rates, we obtain
de x - " "" 1 dm "

__
A = k1CSC X - k2 CX Cx - --Cx (6.78)
dt A B m dt A
dC x
__B = k2C"X "Cx 1 dm "
- --Cx (6.79)
dt A B m dt A
The change of the substrate concentration is

dCs k1 - "
- = ---CsC x (6.80)
dt Yx / s
where YXIS is the yield constant.
Following, Eqs. (6.73), (6.74), (6.78), (6.79), and (6.80) can be solved
" " "
simultaneously to obtain the change of Cx, CXA , CXB , Cs , and m.
1. Cell" division occurs if and only if the structural portion
(mvC xB ) has doubled its initial value, and a dividing cell
apportions each component equally to its two daughter cells.
Therefore, the total number of cells in the system will be
proportional to the structural portion as
n oc mvCx B
(6.81)
The average mass of a cell is equal to the total cell mass divided by
total cell number. Therefore,
Average mass of cell = - oc

m
"
x = ~ mvC (6.82)
C
n mvCx
B
C xB
As a result, this model can also predict the change of the average
cell size with respect to time, which is not possible with the Monod
model. Eqs. (6.73), (6.78), (6.79), and (6.80) can be expressed with the
concentrations in terms of mass per unit culture volume as
Cx = CXA + CXB (6.83)
dC XA =
dt
l/ v -VmvA)k1CSCX - ( VA )k 2CX
mv A
ex
B
(6.84)
dC x
__B = ( -v" )' k 2CX Cx (6.85)
dt mv A R
des
dt
= __ 1_(
YXis V _ mv"
V )k CCx 2 S
(6.86)
where Cx =(mv/V)C x , Cx =(mv/V)C x , Cx =(mv/V)C x ,

A A B B
and Cs = [(V - mv)/VlCs

The mass m is related to C x as
m = CxV (6.87)
Now, the total number of cells is proportional to CXB and the
average mass of a cell to CX/C XB •
Figure 6.23 illustrates simulation curves of a batch culture, which
show the changes of mass (Cx/C xmax ), number (CX/CXB max), and size
of cells (Cx/C XB)' and the substrate concentration (C 5/C 50 ) in
dimensionless form. This curve shows the following features:
1. During a lag phase, cells grows in size but not in number.
2. Cells are largest during the exponential phase.
3. The total cell mass reaches its asymptote before cell number.
4. Cells no longer grow or divide during a stationary phase
1.0 -------------=---~----, 5
0.8 4
0.6 3
0.4 2
0.2 ........................
Size
o 2 4 6 8 10
Time
Fig. 6.23 Simulation results of a batch culture using the two compartment
model proposed by Williams (1967), which show the changes of
mass, number, and size of cells, and the substrate concentration
=
in dimensionless form. Parameters: Cso 1 giL, CXAO 0, Cxao =
= 0.05 giL, k 1 =1.25 Llg hr, k2 =0.0002 Llg hr, v =0.0002 Llg
Even though Williams's model provides many features that
unstructured models are unable to predict, it requires only two
parameters, which is the same number of parameters required for
Monod kinetics.
6.9 NOMENCLATURE
B flow rate of bleeding stream, m 3 I s
C concentration, mass per unit volume of culture, kg/m 3
C mtracellular concentration, mass per unit volume of
biotic phase, kg/m3
c extracellular concentration, mass per unit volume of
abiotic phase, kg/m
constants
cell number density, number of cells/m
dilution rate, S-l
flow rate, m 3 I s
system coefficient for the Monod kinetics, kg/m
flow rate of filtrate stream, m 3 Is
m average mass of cell in a system, kg
m mass of biotic phase, kg
n number of cells
r rate of growth per unit volume, kg/m 3s
r·t,l. rate of jth component formed from ith reaction per unit
volume of a system, intracellular property, kg/m s
r·t, J. rate of jth component formed from ith reaction per unit
volume of a system, extracellular property, kg/ m 3 ~
time, s
doubling time, s
specific volume of a system contajning 0111y biotic phase,
3
m /kg
v working volume of fermenter, In 1
y yield constant
f3 bleeding ratio, defined as Blr
8 average cell division rate, S I
J1 specific growth rate, S·l or kg/m 3s
P density, kg/m3
r residence time, S
SUBSCRIPT
b batch fermenter
input stream
m mixed fermenter
n cell in number basis
X cell in dry weight basis
P product
p plug-flow fermel1ter
5 substrate
6.10 PROBLEMS
6.1 Derive the relationship giving the change with respect to time
of the cell concentration in a batch fermenter, Eq. (6.24).
6.2 Aiba et al. (1968) reported the results of a chemostat study on
the growth of a specific strain of baker's yeast as shown in the
following table. The inlet stream of the chemostat did not
contain any cells or products.
Dilution Inlet cone. (gIL) Steady-state concentration (gIL)
Rate D, hr- 1 glucose CSb Glucose Cs Ehanol Cp Cells Cx
0.084 21.5 0.054 7.97 2.00
0.100 10.9 0.079 4.70 1.20
0.160 21.2 0.138 8.57 2.40
0.198 20.7 0.186 8.44 2.33
0.242 10.8 0.226 4.51 1.25
a. Find the rate equation for cell growth.

b. Find the rate equation for product (ethanol) formation.
6.3 Andrews (1968) proposed the following model for the growth
of microorganisms utilizing inhibitory substrates.
J1 = J1 ma x
1 + K s + Cs
Cs K[
Assume that a chemostat study was performed with a
microorganism. The volume of the fermenter content was 1 L.
The inlet stream was sterile. The flow rate and inlet substrate
concentration were varied and the steady-state concentration of
glucose in the fermenter was measured and recorded as shown
in the table (the data are arbitrary).
a. Determine the kinetic parameters (J1max, Ks, and Ki ) of this
microorganism.
b. If the cell yield, Y XIS' is 0.46 g/ g, what is the steady-state
cell concentration when the flow rate is 0.20 L/h?
c. Andrews concluded in his paper that the primary result of

substrate inhibition in a continuous culture may be process
instability. Explain what might happen if you suddenly
increase the substrate concentration from 30 to 60 giL and
why.
Flow rate Inlet glucose cone. Steady-state glucose cone.
Llhr GSi giL Cs, giL
0.20 30 0.5
0.25 30 0.7
0.35 30 1.1
0.50 30 1.6
0.70 30 3.3
0.80 30 10
0.50 60 30
0.60 60 22
0.70 60 15
6.4 Rate equations for the cells (yeast), substrate in the ethanol
fermentation process are given (glucose), and product as
follows:
n
r = dC x - Cs 1- Cp C
x dt - Jimax ( K +C )( Cpm ) x
s s
dCs 1 dC
rs = --=-----x
-
dt Yx / P dt
dC p x1 dC
rp= - - = - - - - -
dt Yx / P dt
where K s = 1.6giL, J.1max = 0.24 hr-1, YX/P = 0.06, YXIS = 0.06,
C pm = 100 giL, Cpo = 0, Cxo = 0.1 giL, and n = 2.
a. Calculate the change of Cx , Cp' and Cs as a function of time
when Cs = 001 giL.
b. Show the effect of the initial substrate concentration on the
Cx versus t curve.
c. Show the effect of the maximum growth rate (Pmax) on the
Cx versus t curve (C 51 = 10(}g/L).
6.5 The growth rate of E. coil in synthetic medium can be expressed
by Monod kinetics as
0.935C C
s x
rx = ---"--- [g/Lhr]
0.71 + Cs
where Cs is the concentration of a limiting substrate, glucose.

You are going to cultivate E. coli in a steady-state CSTF
(working volume: 10 L) with a flow rate of 7 L/hr. The initial
substrate concentration is 10 giL and the cell yield constant
(Y XIS) is 0.6. The feed stream is sterile.
a. What will be the doubling time and the division rate of the
cells in the CSTF?
b. What will be the cell and substrate concentrations of the
outlet stream?
c. If you connect one more 10/L CSTF to the first one, what
will be the cell and substrate concentrations in the second
fermenter?
d. If you increase the flow rate from 7 to 10 L/hr for these two
fermenters connected in series, what will happen and why?
Make a recommendation to avoid the problem if there is
any.
6.6 Suppose that the growth rate of a microorganism can be
expressed as the following equation:
rx = J.lmax [1- exp(-CsIKslC x
where J.1max = 1.365 hr-1 and K s = 6.8 giL. The cell yield YXIS is
found to be 0.45.
a. If you cultivate this microorganism in a 10 L CSTF with the
flow rate of 2.8 L/hr, what will be the steady-state cell
concentration of the outlet stream? The substrate
concentration of the inlet stream is 13g/L. The inlet stream
is sterile.
b. Explain the difference between this model and Monod
model by using /l versus Cs graph.
6.7 Herbert et al.(1956) reported that the growth kinetics of
Aerobacter cloacae in a chemically defined medium (glycerol as a
limiting substrate) could be expressed by Monod kinetics as
follows:
dC
rx = - -x=
PmaxCSCX
dt Ks+C s
where J-lnlax = 0.85 hr- and Ks = 1.23 X 10-2 giL. The yield was
1
found to be 0.53 g dry weight of organism/ g glycerol used.

You are a biochemical engineer who has been assigned the task
of designing the most effective continuous fermentation system
to grow the microorganism (Aerobacter cloacae] with glycerol as
its limiting substrate. For the followiD-g three questions, the
concentration of glycerol in the feed stream and that of glycerol
in the outlet stream should be 3 giL and 0.1 giL, respectively.
a. Since you have learned that the llr x versus Cs curve for
Monod kinetics has a U shape, you have recommended that
the most effective system would be the combination of a
continuous stirred-tank fermenter (CSTF) and a plug-flow
fermenter (PFF). You were quite sure of this because the
substrate concentration in the outlet stream has to be so
low. However, your boss is insisting that the use of second
PFF in addition to the first CSTF will not improve the
productivity very much. Who is right? Prove whether you
are right or wrong by drawing the 1/rxversus C s curve for
this microorganism. Does it have a U shape? Discuss why
you are right or wrong. (If you are right, think about how
you can nicely correct you boss's wrong idea. If you are
wrong, it will teach you that you have to be careful not to
make a quick conclusion without adequate analysis.)
b. Recommend the best fermenter system (fermenter type and
volume) which can handle 100 Llhr of feed stream. The
best fermenter system is defined as that which can produce
the maximum amount of cells per unit time and volume.
c. If Ks = 1.23 giL instead of 1.23 x 10-2 giL, what is the best
fermenter system (fermenter type, volume) which can
handle 100 Llhr in the feed stream. Draw the block
diagram of the fermenter system with the concentrations of
the substrate and the cells in the inlet and outlet streams of
each fermenter. How is this case different from the case of
part (a) and why?
6.8 Suppose you have an organism that obeys the Monod equation:
dC- )1max Cs CX
x =-
- _............-
dt Ks +Cs
1
where )1max = 0.5,hr- and Ks = 1 giL.
The organism is being cultivated in a steady-state CSTF, where
F = 100 L/hr, CSi = 50 giL, and Y XIS = 0.5.
a. What size vessel will give the maximum total rate of cell
production?
b. What are the substrate and cell concentrations of the
optimum fermenter in part (a)?
c. If the exiting flow from the fermenter in part (a) is fed to a
second fermenter (CSTF), what should be the size of the
second fermenter to reduce the substrate concentration to 1
giL?
d. If the exiting flow from the first fermenter in part (a) is fed
to a second fermenter whose size is the same as the first,
what will be the cell and substrate concentrations leaving
the second fermenter?
6.9 You are going to cultivate yeast, Saccharomyces cerevisiae, by

using a 10 m -fermenter your company already owns. You want
to find out the amount of ethanol the fermenter can produce.
Therefore, a chemostat study was carried out and the Monod
kinetic parameters for the microorganism grown in the glucose
medium at 30°C, pH 4.8, were found to be: Ks = 0.0025 giL and
Jlmax = 0.25 h -1. The ethanol yield (YPIS) is 0.44 (gl g) and cell
yield (Y XIS) is 0.019 (gl g). The inlet substrate concentration is 50
giL.
a. What flow rate will give the maximum total ethanol
production in the continuous fermenter and what is the
maximum ethanol production rate?
b. If you want to convert 95 percent of the incoming substrate,
what must the ethanol production rate be for the
continuous fermenter?
c. If you have two 5 m 3-fermenters instead of one 10 m 3_
fermenter, what is your recommendation for the use of
these fermenters to convert 95 percent of the incoming
substrate? Would you recommend connecting two
fermenters in series to improve the productivity? Why or
why not?
6.10 You are a biochemical engineer in a pharmaceutical company.
Your company is a major producer of penicillin. Currently,
what kind of fermenter is your company using for penicillin
production? Why?
Your boss asked you to study the possibility of using an air-lift
fermenter as a replacement since it has many advantages. What
is your recommendation?
6.11 Consider an organism with the following data for a 1-L
chemostat, using an inlet substrate concentration Csi of 30 giL:
Flow rate Concentration (giL)
(mLlhr) Substrate Cell Products
F Cs ex Cp
27.5 10.0 12.0 1.04
24.2 5.56 14.7 1.27
22.1 3.70 15.8 1.37
18.8 2.32 16.7 1.44
a. In a continuous, perfectly mixed vessel at steady state with
no cell death, if inlet substrate concentration CSi now
equals 25.0 giL and cell concentration C Xi is 0.0 giL, what
dilution rate D will give the maximum total rate of cell
production? What are the outlet cell and substrate
concentrations at this dilution rate?
b. Using the preceding organism, your boss would like you to

design a continuous reactor system with an inlet flow and
substrate concentration of 250 Llhr and 25 giL,
respectively, which will produce an overall yield of
product of 2100 kglyr, given an operating time of 300
day Iyr at 24 hr I day. Assume no cells or product in the
system inlet. Would you recommend a single fermenter of
two fermenters in series? Design a system which will meet
production constraints while minimizing total fermenter
volume. Report reactor volumes and effluent cell, substrate,
and product concentrations for the proposed fermenter(s).
[Contributed by Brian S. Hooker, TriState University.]
6.12 A strain of yeast is being cultivated in a 30-L CSTF with a cell
recycling system (cell settler) as shown in the following figure.
The cell settler was designed so that the cell concentration of its
outlet stream is 30 percent of that of its inlet stream, whereas
the substrate concentrations of the two streams are the same.
The growth rate of the cells can be represented by the Monod
kinetics with the parameters: K s = 0.05 giL, J.1max = 0.3 h-1 , and
YXIS = 0.025. Calculate the steady-state substrate and cell
concentrations in the fermenter. The inlet substrate
concentration is 100 giL and the flow rate is 20 L/hr. The feed
stream is sterile.
F F
CSt' 03C
. x
Cs
Cs
6.13 You need to cultivate hypothetical microbial cells with the
Monod kinetic parameter values of J.1max = 5.0 hr-1 and K s = 20
giL. The cell yield (YXIS) is 0.4 and the substrate concentration
is 30 giL. The required substrate conversion is 97%. Estimate
the residence time required for the following fermenter
configurations
a. Plug-flow fermenter with the inlet cell concentration of 0.5
giL.
b. Plug-flow fermenter with a recycle stream (no cell
separator) with the flow rate of 0.4F. The inlet cell
concentration is 0.5 giL. How your answer will be changed
if you reduce the inlet cell concentration to zero?
c. One steady-state CSTF with a recycle stream (no cell

separator) with the flow rate of 0.4F. The inlet stream is
sterile.
6.14 By using the structured model proposed by Ramkrishna et ale
(1967), show the change of the concentrations [in g dry weightl
L] of G-mass, H-mass, and inhibitor, and the fraction of G-mass
with time during a batch cultivation of a microorganism which
has the following parameters and initial conditions:
)1 = 0.5 hr-1 as' = 2
Ji/ = 2.5 hr- 1
aT = a
K s = 0.2 giL aT' 0.2 x 10-4
s
K = 0.1 giL aT1 = 0.0267
K = 150 LI g hr aT1' = 0
K' = 70 Llg hr C S1 = 10 giL
5
KG = 3.0 * 10- giL CT1 = a giL
KG' = 0.5 X 10-5 giL CXH1 = 8.0 X 10-6 giL
as = 8 C XG1 = 1.0 X 10-6 giL
Compare your simulation result with Figure 14 of the paper by
Ramkrishna et al. (1967). If you showed the change of the cell
concentrations in g dry weightlliter, the shape of the curves are
quite different from those in the paper. What are the
differences? Explain. Do the parameter values predict realistic
growth curves? What new features can this model predict
which the Monod model cannot?
6.11 REFERENCES
Advanced Continuous Simulation Language: User Guide Reference Manual.
Concord, MA: Mitchell and Gauthier, Assoc., Inc., 1975.
Aiba, 5., A. E. Humphrey, and N. F. Millis, Biochemical Engineering (2nd ed.),
p. 304. Tokyo, Japan: University of Tokyo Press, 1973.
Aiba, 5., M. Shoda, and M. Nagatani, "Kinetics of Product Inhibition in
Alcohol Fermentation," Biotechnol. Bioeng. 10 (1968):845-864.
Andrews, J. F., "A Mathematical Model for the Continuous Culture of
Microorganisms Utilizing Inhibitory Substrates," Biotechnol. Bioeng.
10(1968):707-723.
Bowen, R. L., "Unraveling the Mysteries of Shear-sensitive Mixing Systems,"
Chern. Eng. 9 (1986):55-63.
Carberry, J. J., Chemical and Catalytic Reaction Engineering, p. 52. New York,
NY: McGraw-Hill Book Co., 1976.
Feder, J.and W. R.Tolbert, "The Large-Scale Cultivation of Mammalian
Cells," Scientific American 248 (1983):36-43.
Fredrickson, A. G., "Formulation of Structured Growth Models," Biotech.

Bioeng. 18 (1976):1481-1486.
Harder, A.and J. A. Roels, "Application of Simple Structured Models in
Bioengineering," Adv. Biochem. Eng. 21 (1982):56-107.
Herbert, D., R. Elsworth, and R. C. Tellmg,"The Continuous Culture of
Bacteria; a Theoretical and Experimental Study," .j. Gen. Microbiol. 14
(1956):601-622.
Hill, G. A. and C. W. Robinson, "Minimum Tank Volumes for CFST
Bioreactors in Series," Can. J. Chern. Eng. 67 (1989):818-824.
Hooker, B. S., J. M. Lee, and G. An, "Cultivation of Plant Cells in a Stirred
Vessel: Effect of Impeller Design," Biotech. Bioeng. 35 (1990):296-304.
Levenspiel, 0., Chemical Reaction Engineering (2nd ed.), p. 150. New York, NY:
John Wiley & Sons, 1972.
Luedeking, R., "Fermentation Process Kinetics," in Biochemical and Biological
Engineering Science, ed.N.Blakebrough. London, England: Academic
Press, Inc., 1967, pp. 181-243.
Monod,J., "The Growth of Bacterial Cultures," Ann. Rev. Microbiol 3
(1949):371-394.
Moo-Young, M., G.Van Dedem, and A. Bmder,"Design of Scraped Tubular
Fermentors," Biotech. Bioeng. 21 (1979):593-607.
Ramkrishna, D., A. G. Fredrickson, and H. M. Tsuchiya,"Dynamics of
Microbial Propagation: Models Considering Inhibitors and Variable Cell
Composition," Biotech. Bioeng. 9 (1967):129-170.
Russell,!. W. F., 1. J. Dunn, and H. W. Blancl1, "The Tubular Loop Batch
Fermentor: Basic Concepts," Biotech. Bioeng. 16 (1974): 1261-1272.
Schiigerl, K., "New Bioreactors for Aerobic Processes," Int. Chern. Eng.
22(1982):591-610.
Tsuchiya, H. M., A. G. Fredrickson, and R. Aris, "Dynamics of Microbial Cell
Populations," in Adv. Chern. Eng., vol. 6, eds.T.E.Drew, J.\V.Hoopes, Jr.,
and T.Vermeulen. New York, NY: Academic Press, 1966, pp. 125-206.
Williams, F. M., "A Model of Cell Growth Dynamics," J. Theoret. BioI.
15(1967):190-207.

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6

Cell Kinetics and Fermenter Design

Table 6.1 Various Models for Cell Kinetics

Besides the assumptions for the cells, the medium is formulated so

6.3 GROWTH CYCLE FOR BATCH CULTIVATION

6.3.1 Lag Phase

6.3.2 Exponential Growth Phase

6.3.3 Factors Affecting the Specific Growth Rate

Fig. 6.2 Dependence of the specific growth rate on the

Several other models have been proposed to improve the Monod

Ji = Jimax[l- exp(-Cs/Ksl (6.13)

If several substances can limit the growth of a microorganism, the

The preceding equations both describe the product inhibition

6.3.4 Stationary and Death Phase

The disadvantage of the STF stems from its advantages. The

Fig. 6.3 Cutaway diagram of a fermenter used for penicillin production.

or a pH controller. The temperature is controlled by heating or

6.4.1 Batch or Plug-Flow Fermenter

It should be noted that Eq. (6.21) only applies when rx is larger

curve is a more straightforward way to determine it. However, the

Fig. 6.4 Schematic diagram of (a) batch stirred-tank fermenter and

Eg. (6.22) can be integrated if we know the relationship between Cs

Substitution of Eq. (6.23) into Eq. (6.22) and integration of the

Fig. 6.5 A graphical representation of the batch growth time t - t1

The Monod kinetic parameters, Ilmax and K s, cannot be estimated

while in the Monod equation,

Fig. 6.6 Solution to Example 6.1.

6.4.2 Ideal Con tin uous Stirred-tank Fermen ter

reservoir of sterile medium. Once growth has been initiated, fresh

Fig. 6.7 Schematic diagram of continuous stirred-tank fermenter (CSTF)

Continuous culture systems can be operated as chemostat or as

where D is known as dilution rate and is equal to the reciprocal of

D = f.l = ~ = f.lmaxCs (6.30)

From Eq. (6.30), Cs can be calculated with a known residence time

It should be noted, however, that the preceding expression is only

Cp = CPi + YPIS (C Si - Ks ) (6.34)

6.4.3. Evaluation of Monod Kinetic Parameters

J1 = Jlmax - K s CJ1 (6.37)

a. Find the rate equation for cell growth.

o 0.5 1.0 1.5 2.0

6.4.4 Productivity of CSTF

The operating condition for the maximum productivity of the

Fig. 6.10 The change of the concentrations of cells and substrate

The productivity is maximum when drxldC x = o. After substituting

a= ~Ks +CSi (6.40)

6.4.5 Comparison of Batch and CSTF

where to is the time required to reach an exponential growth phase.

Fig. 6.12 Graphical illustration of the residence time required

Fig. 6.13 Graphical illustration of the total residence time required

6.5 MULTIPLE FERMENTERS CONNECTED IN SERIES

6.5.1 CSTF and PFF in Series

Fig. 6.14 Schematic diagram of the two fermenters,

CPt = CPi + Y p/ s (CSi - K

For the second PFF, the residence time can be estimated by

Since growth yield can be expressed as

_( KsYx/s ) CX2 KsYx / s CS1 (6.49)

Eqs. (6.48) and (6.49) have to be so~ved simultaneously to estimate

6.5.2 Multiple CS TFs in Series

Fig. 6.15 Schematic diagram of the multiple CSTFs connected in series.

By solving Eqs. (6.50), (6.51), and (6.52) simultaneously, we can

Ks =0.71 gIL, Yx/s=0.6, CSi =10g/L,Cxi = O.}

D =£ = Ilmax Cs = 0.7(5) = 0.35

Therefore, from Eq. (6.39) through Eq. (6.42),